Many assays for detecting toxic molecules in effluents are inefficient when these molecules are present at very low concentration. Assays that are efficient for this purpose such as those made with living organisms and cells like algaes, daphneas, trouts, hepatocytes, or worms are time consuming (up to 72 hours). These methods investigate the presence of toxic molecules in water by measuring the survival of these organisms and cells in contact of the water being tested.
Assays using the response of photosynthetic organs to toxic molecules have been recently devised which in addition to reducing the response time of the assay to 10 minutes are advantageously sensitive to molecules affecting photosynthetic electron transport and simple to use. Thylakoids are the organs responsible for photosynthesis in phytoplankton, algaes and higher plants. In these assays, the physiological response of thylakoids to stress such as toxic molecules is assessed through indirect measurement of photosynthesis, based on a functional relationship between photosynthetic efficiency and a stimulated fluorescence signal. Fluorescence emission by these organs is due to their ability to use light for photosynthesis. Certain methods determine photosynthetic activities from isolated Photosystem II (PS II). For instance, EP patent no. 1,134,585 published on Sep. 19, 2001 in the name of Giardi et al. describes the use of PS II isolated from thylakoids membranes to monitor herbicides. Other methods determine photosynthetic activities of whole plants. For instance, U.S. Pat. No. 6,121,053 issued on Sep. 19, 2000 in the name of Kolber et al. describes a pulsed fluorometer permitting the measurement of photosynthesis efficiency in plants, phytoplankton and algaes. In Laberge (1999) and (2000) are described thylakoids chemically immobilised in a BSA-glutaraldehyde matrix or encapsulated in poly(vinylalcohol) bearing styrylpyridinium substituent groups, respectively.
None of these systems have been demonstrated to be stable at room temperature.
Other methods have used thylakoid membranes themselves which also degrade disadvantageously rapidly at room temperature. These thylakoids samples must be kept at 4° C. and even then only survive a few hours (Table 2). The need to keep the isolated thylakoid membranes at low temperature until immediately before the assay makes their use in in situ assays difficult.
An object of the present invention is therefore to provide improved photosynthesis-based biosensors and bioassays for detecting toxic molecules in fluids, and methods and kits for using same.